Resident enteric flora modulates the development of colitis in gnotobiotic mice

This dissertation provides in depth studies on how the composition of the resident intestinal microflora modulates the development of colitis in gnotobiotic mice. It is arranged into 4 sections including: (1) General introduction---this includes an overview of human inflammatory bowel disease (IBD) and a literature review of the host-microbial interactions contributing to IBD in humans and in experimental animals. In brief, this section provides strong scientific evidence that luminal bacteria are an important environmental factor in the development of intestinal inflammation in humans and animal models; and that bacterial species vary in their ability to incite mucosal inflammation in a susceptible host. This section also introduces our use of a novel gnotobiotic mouse model of intestinal inflammation utilized in the two attached research publications. (2) Paper 1---entitled Composition of the resident enteric flora modulates colitis in defined flora mice characterizes antigen-specific immune responses to a defined gastrointestinal flora in the pathogenesis of experimental colitis. Gnotobiotic mice possessing an altered schaedler flora were used to evaluate the host\u27s response following challenge with H. bilis and/or B. hyodysenteriae. These data indicate that the nature of the gastrointestinal flora significantly influences the immunologic bias of the immune response and the presence of Helicobacter species do not apriori predispose toward a Th1 immune response. (3) Paper 2---entitled Gnotobiotic mice colonized with Helicobacter bilis demonstrate antigen-specific immune responses to resident enteric flora investigates the kinetics of host responses to resident enteric flora following colonization with H. bilis. These data indicate that immunologic responses of gnotobiotic C3H mice colonized with H. bilis developed rapidly, persisted over the 10 week study period and were of mixed Th1:Th2 phenotype. Furthermore, we propose a potential pathogenic role for H. bilis in perturbing the normal resident microflora and inducing antigen-specific immune responses against multiple resident bacterial species which may predispose to colitis. (4) General Conclusions---provides an overall summary of the conclusions drawn from papers 1 and 2 and proposes future studies regarding host-microbial interactions mediating chronic intestinal inflammation

Gastrointestinal Endoscopic Exfoliative Cytology: Techniques and Clinical Application

Cytologic examination of exfoliative specimens obtained during endoscopy is a useful and reliable adjunct to mucosal biopsy for the diagnosis of gastrointestinal (GI) tract diseases in dogs and cats. Clinical advantages of endoscopic cytology include simplicity, rapidity of diagnosis and minimal invasiveness. Cytologic smears are graded on the basis of objective criteria, including the presence and number of inflammatory, atypical, and epithelial cells as well as the presence of bacteria, hemorrhage, debris/ingesta, and mucus. There is high correlation between results obtained from endoscopic cytology and histologic examination, and discordant results are infrequent. Brush cytology is useful in detecting mucosal inflammation, whereas touch cytology is more likely to detect acute purulent and erosive mucosal lesions. Alimentary lymphoma my be readily diagnosed using either technique. This article provides an overview of how cytologic smears are prepared and evaluates their diagnostic accuracy

Genome-wide association studies of inflammatory bowel disease in German shepherd dogs

Canine Inflammatory Bowel Disease (IBD) is considered a multifactorial disease caused by complex interactions between the intestinal immune system, intestinal microbiota and environmental factors in genetically susceptible individuals. Although IBD can affect any breed, German shepherd dogs (GSD) in the UK are at increased risk of developing the disease. Based on previous evidence, the aim of the present study was to identify single nucleotide polymorphisms (SNPs), which may confer genetic susceptibility or resistance to IBD using a genome-wide association study (GWAS). Genomic DNA was extracted from EDTA blood or saliva samples of 96 cases and 98 controls. Genotyping of cases and controls was performed on the Canine Illumina HD SNP array and data generated was analyzed using PLINK. Several SNPs and regions on chromosomes 7,9,11 and 13 were detected to be associated with IBD using different SNP-by-SNP association methods and FST windows approach. Searching one Mb up-and down-stream of the most significant SNPs, as identified by single SNP analysis as well as 200Kb before and after the start and the end position of the associated regions identified by FST windows approach, we identified 63 genes. Using a combination of pathways analysis and a list of genes that have been reported to be involved in human IBD, we identified 16 candidate genes potentially associated with IBD in GSD

Combined effects of chemotherapy and Nrf2 activation in colorectal cancer cells in vitro

Colon cancer is the third leading type of cancer diagnosis in the United States (Siegel et al, 2017). Common treatments include chemotherapy, which can be toxic to the patient and produce multiple adverse side effects(Sarkar, 2008). Combination therapies with chemotherapy drugs and other compounds have been reported to decrease tumor growth in breast and colon cancer by increasing efficacy of chemotherapeutic agents at lower doses, thus reducing off-target adverse effects(Borcherding et al, 2015; Chen et al 2017). Both activation of Nrf2, a transcription factor that induces expression of anti-oxidant genes, and dopamine receptor agonists, have been shown to reduce tumor growth in multiple cancer types (Borcherding et al, 2015; Melba et al, 2013). Thus, we examined whether combining a common chemotherapy drug, Doxorubicin, with a Nrf2 activator, CDDO-ME, or a dopamine-type-1 receptor agonist, Fenoldopam, improved efficacy of chemotherapy. Treatment of HT29 and HCT116 colorectal cancer cells in vitro with or CDDO-ME in conjunction with Doxorubicin augmented the effects of Doxorubicin alone, as determined by MTT assay. The results support that Doxorubicin had an effect on both cell lines above concentrations of 100 nM. However, Fenoldopam, a dopamine-type-1 receptor agonist, did not significantly affect cell viability. Therefore, the effects of Doxorubicin may be achieved at a lower dose when administered with CDDO-ME

Effects of Six Common Dietary Nutrients on Murine Intestinal Organoid Growth

The intestinal epithelium of the gastrointestinal (GI) tract constantly renews itself to absorb nutrients and provide protection for the body from the outside world. Since the intestinal epithelium is constantly exposed to various chemicals and dietary components, it is critical to determine which constituents promote or inhibit intestinal epithelium health and growth rate. Intestinal organoids, three-dimensional miniature models of the intestines, represent an ex vivo tool to investigate intestinal physiology and growth patterns. In this study, we measured the growth rates of murine intestinal organoids exposed to various concentrations of different dietary constituents. Results indicate that caffeic acid inhibited organoid growth in a concentration-dependent manner, curcumin exhibited variable effectiveness, and vitamin C had no effect on organoid growth

Intestinal Stem Cells to Advance Drug Development, Precision, and Regenerative Medicine: A Paradigm Shift in Translational Research

Recent advances in our understanding of the intestinal stem cell niche and the role of key signaling pathways on cell growth and maintenance have allowed the development of fully differentiated epithelial cells in 3D organoids. Stem cell-derived organoids carry significant levels of proteins that are natively expressed in the gut and have important roles in drug transport and metabolism. They are, therefore, particularly relevant to study the gastrointestinal (GI) absorption of oral medications. In addition, organoids have the potential to serve as a robust preclinical model for demonstrating the effectiveness of new drugs more rapidly, with more certainty, and at lower costs compared with live animal studies. Importantly, because they are derived from individuals with different genotypes, environmental risk factors and drug sensitivity profiles, organoids are a highly relevant screening system for personalized therapy in both human and veterinary medicine. Lastly, and in the context of patient-specific congenital diseases, orthotopic transplantation of engineered organoids could repair and/or replace damaged epithelial tissues reported in various GI diseases, such as inflammatory bowel disease, cystic fibrosis, and tuft enteropathy. Ongoing translational research on organoids derived from dogs with naturally occurring digestive disorders has the potential to improve the predictability of preclinical models used for optimizing the therapeutic management of severe chronic enteropathies in human patients

A Clinical Index for Disease Activity in Cats with Chronic Enteropathy

Background: There is a need for a clinically useful, quantitative index for measurement of disease activity in cats with chronic enteropathy (CE). Objective: To develop a numerical activity index that is of practical value to clinicians treating CE in cats. Animals: Eighty-two cats with CE. Methods: Retrospective case review of 59 cats diagnosed with inflammatory bowel disease (IBD). Prospective validation study of 23 cats having either IBD or food-responsive enteropathy (FRE). Multivariate regression analysis was used to identify which combination of clinical and laboratory variables were best associated with intestinal inflammation of IBD. This combination of variables was expressed in a score that was used as an activity index for the prospective assessment of disease activity and of the effect of treatment in cats with IBD or FRE. Results: The combination of gastrointestinal signs, endoscopic abnormalities, serum total protein, serum alanine transaminase/alkaline phosphatase activity, and serum phosphorous concentration had the best correlation with histopathologic inflammation and comprise the feline chronic enteropathy activity index (FCEAI). Positive treatment responses in cats with CE were accompanied by significant (P \u3c .05) reductions in FCEAI scores after treatment. Conclusions and Clinical Importance: The FCEAI is a simple numerical measure of inflammatory activity in cats with CE. The scoring index can be reliably used in the initial assessment of disease severity for both IBD and FRE and as a measure of clinical response to treatment for these disorders